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OPEN Evaluation of the efect of GM-CSF blocking on the phenotype and function of human monocytes Noushin Lotf1,2, Guang-Xian Zhang1, Nafseh Esmaeil2* & Abdolmohamad Rostami1*

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent that prompts the proliferation of bone marrow-derived macrophages and granulocytes. In addition to its efects as a , GM-CSF plays an important role in chronic infammatory autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Reports have identifed monocytes as the primary target of GM-CSF; however, its efect on monocyte activation has been under-estimated. Here, using fow cytometry and ELISA we show that GM-CSF induces an infammatory profle in human monocytes, which includes an upregulated expression of HLA-DR and CD86 molecules and increased production of TNF-α and IL-1β. Conversely, blockage of endogenous GM-CSF with antibody treatment not only inhibited the infammatory profle of these cells, but also induced an immunomodulatory one, as shown by increased IL-10 production by monocytes. Further analysis with qPCR, fow cytometry and ELISA experiments revealed that GM-CSF blockage in monocytes stimulated production of the CXCL-11, which suppressed proliferation. Blockade of CXCL-11 abrogated anti-GM-CSF treatment and induced infammatory monocytes. Our fndings show that anti-GM-CSF treatment induces modulatory monocytes that act in a CXCL-11-dependent manner, a mechanism that can be used in the development of novel approaches to treat chronic infammatory autoimmune diseases.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine that stimulates the proliferation of bone marrow-derived macrophages and granulocytes. Various cell types produce this cytokine, including activated T cells, monocytes/macrophages, B cells, NK cells, endothelial, epithelial, and fbroblasts cells1. GM-CSF has been identifed as a major cytokine in chronic infammatory autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA)2,3 GM-CSF plays a crucial role in RA progression and aug- ments infammatory immune responses in synovia4,5. Moreover, GM-CSF-producing CD4+ T cells in the blood and lesions of untreated MS patients correlate with disease severity6. We have shown that GM-CSF is necessary for the pathogenicity of T17 cells in experimental autoimmune encephalomyelitis, the prototypical animal model for MS7. GM-CSF exerts its function by binding to its receptor, which is composed of two diferent subunit α (CD116; GM-CSF Rα) and β chains (CD131; GM-CSF Rβ) with low and high afnity, respectively. Te alpha subunit is involved in -specifc binding while the beta chain plays a central role in the signal transduction pathway8. GM-CSF signaling afects the survival and activation of myeloid cells, dendritic cell (DC) diferentiation and M1 macrophage phenotype polarization; it boosts antigen presentation, induces phagocytosis, recruits monocytes and other myeloid populations from bone marrow to circulation and promotes chemotaxis9,10. It has been recently demonstrated that CCR2+Ly6Chi infammatory monocytes are a target of GM-CSF in CNS autoimmunity by stimulating infammatory monocytes and their conversion into pathogenic macrophage-derived dendritic cells11–13. GM-CSF-activated monocytes migrate across the blood-brain barrier (BBB) and mediate BBB rupture by increasing expression of the endothelial adhesion molecules ICAM-1 and VCAM-114,15. GM-CSF also induces CCR2 expression in monocytes, which gives them an increased ability to cross the BBB. In EAE and MS, the CCR2-CCL2 axis has been previously shown to be a signifcant driver of infammatory leukocyte infltration into the CNS, and its activity positively correlates with disease pathogenesis16–18. Migration of leukocytes into the CNS is also mediated by CXCL9 and CXCL10 produced by glial cells19. Activated T lymphocytes in MS patients express CXCR3, which is the corresponding receptor of CXCL9, CXCL10, and CXCL-11 chemokines20.

1Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA. 2Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. *email: [email protected]; [email protected]

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It has been previously shown that while CXCL9 is a homing chemokine in the CNS, CXCL10, and CXCL-11 are induced afer infammation, and their role in infammation is less clear21–23. CXCL10 is involved in intrathecal infammation24. Interestingly, CXCL-11 is upregulated in MS patients afer IFN-β therapy and the decrease in the number of relapses may be linked to the increase in CXCR3 ligands in the serum of IFN-β-treated MS patients25. In this study, we analyzed the efect of GM-CSF on the phenotype and function of human monocytes. We found that GM-CSF treatment induces an infammatory phenotype in monocytes, while endogenous GM-CSF blocking is accompanied by an immunomodulatory phenotype. Further, GM-CSF blockade promoted CXCL-11 expression, and recombinant CXCL-11 inhibited the GM-CSF-induced proinfammatory impact of monocytes on T cells. Our fndings show that one of the mechanisms by which GM-CSF induces infammatory monocytes is the inhibition of CXCL-11 production and that this chemokine may be harnessed to suppress deleterious infam- matory responses observed in chronic infammatory diseases such as MS. Methods Isolation of human monocytes and culture treatments. All subjects gave informed consent before their participation in the current study. All human studies were approved by the Institutional Review Board (IRB) of Tomas Jeferson University, and all methods were performed in accordance with the relevant guidelines and regulations. Whole blood samples were collected from healthy donors and peripheral blood mononuclear cells (PBMCs) were enriched by gradient centrifugation in Ficoll. CD14+ monocytes were isolated by positive selection using magnetic beads following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Te purity of cells was above 90%, measured by fow cytometry. Monocytes were seeded (1 × 106/ml) in 24-well plates and cultured in Iscove’s Modifed Dulbecco’s Medium (IMDM) (Gibco, Gaithersburg, MD, USA) supple- mented with 10% FBS, 1% penicillin/streptomycin antibiotic (Gibco), 2 mM glutamine and 2β- Mercaptoethanol (50 µg/ml, Gibco). Monocytes were activated with lipopolysaccharide (100 ng/mL, Sigma-Aldrich) for 18 h at 37 °C in the presence of recombinant human GM-CSF (10 ng/mL, R&D Systems, Minneapolis, MN, USA) or anti- GM-CSF (10 µg/mL, Biolegend, San Diego, CA). LPS-activated cells (mature monocytes) cultured with PBS were used as controls and culture of monocytes without LPS stimulation were considered as immature cells.

RNA extraction, cDNA synthesis, and qPCR array. RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and the RNA concentration and quality were determined with Nanodrop (Thermofisher Scientific, Waltham, MA, USA). cDNA synthesis was performed from 1 µg of RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using the TaqMan Array Human Immune Response (Applied Biosystems). Real-time PCR for CXCL-11 (Hs03003631_g1) was conducted™ according to the manufacturer’s instructions using TaqMan reagents (TermoFisher). Relative expression was calculated following the 2-ΔΔCT method, where 18 s (Hs03003631_g1) was considered the housekeeping gene.

Flow cytometric analysis. For assessment of surface and intracellular cytokine expression, monocytes were collected afer 24 hours and stimulated for three hours with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO,USA), 500 ng/ml ionomycin (Sigma-Aldrich), and 1 µg/ml GolgiPlug (BD Biosciences, San Jose, CA, USA). Cells were stained with anti-CD14 (M5E2, Biolegend), anti-CD16 (3G8, Biolegend.), anti-CD11b (ICRF44, Biolegend), anti-HLA-DR (L243, Biolegend), anti-CD80 (2D10, Biolegend), anti-CD86 (IT2.2, Biolegend), anti-CD83 (HB15e, Biolegend) and anti-PDL1(29E.2A3 Biolegend). Surface staining was performed for 20 min at 4 °C in the dark, and afer washing cells were fxed using 100 µl/tube fxation bufer at room temperature for 30 min (Termofsher Scientifc). Subsequently, the monocytes were permeabilized with 100 µl/tube Permeabilization Bufer (Termofsher Scientifc) and then stained with fuorochrome-conjugated antibodies for intracellular markers including anti-IL-10 (JES3-9D7, Biolegend), anti-TNF-α (MAb11, Biolegend) and anti-IL-1β (H1b-98, Biolegend), anti-IL-27 (B032F6, Biolegend) overnight at 4 °C in the dark. Also, the cells harvested from T- cells × monocytes co-culture treatments were stained for surface and intra- cellular markers with fuorochrome-conjugated antibodies including anti-CD3(SK7, Termofsher Scientifc), anti-CD4 (OKT4, Biolegend) anti-PDL-1 (29E.2A3, Biolegend), anti-IL-10 (JES3-19F1, Biolegend), anti-IFN-γ (B27, Biolegend), anti-RORɤt (Q21-559, BD Biosciense), anti-CD39 (A1, Biolegend). Samples were acquired on a BD FACS Aria Fusion (BD Biosciences) fow cytometry instrument and data ana- lyzed using Flowjo sofware 10. Te instrument calibration was examined before running the samples with BD FACSDiva™ CS&T research beads (CS&T research beads, BD Biosciences). Co-culture experiments. To examine the effects of GM-CSF, CXCL11 and their blockade on T cell responses, human monocytes and naïve T cells were isolated from peripheral blood of healthy volunteers by magnetic cell isolation according to the company’s instructions (Miltenyi Biotec). Purifed monocytes at 2 × 104 were seeded to U bottom 96 well plates and cultured in IMDM culture medium, which contained 10% FBS and 1% penicillin-streptomycin, in 7 groups with diferent treatment conditions including PBS, GM-CSF (10 ng/ml), Anti-GM-CSF (10 µg/ml), RHCXCL11(15 ng/ml) (R&D Systems), RHCXCL11 + GM-CSF, Anti CXCL11 (10 µg/ ml) (R&D Systems, USA), and Anti-GM-CSF + Anti-CXCL11 for 24 hours. Te following day, isolated naïve T cells were labeled with Cell Trace Violet (Termo Fisher) following the manufacturer’s instructions. T cells were then stimulated with the soluble anti-CD3 antibody at a concentration of 1 µg/ml. T cells were then added (1 × 105 cells per well) to the same monocyte wells for cell-cell interactions and kept humidifed in a 5% CO2 incubator at 37 °C for 72 hours. Afer that time, cell culture supernatant was carefully removed and immediately frozen at −20 °C. To measure the proliferative capacity of T cells, CFSE intensity in the cells was assessed by fow cytometry.

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Figure 1. Overexpression of MHCII and costimulatory molecules in GM-CSF treated human monocytes. (A) Graphs summarize fow cytometry fndings. (B) Results are reported as mean ± SEM from three independent experiments.

Enzyme-linked immunosorbent assay (ELISA). Supernatants from cultures described above were stored at −20°C until used to detect CXCL-11, IL-10, IL-1β, TNF-α, IL-27, and IFN-γ by ELISA kits following the manufacturer’s recommendations (R&D Systems).

Statistical analysis. Statistical analyses were done using Graph Pad Prism version 7. Comparison between groups was performed using Student’s t-test (two groups) and one-way ANOVA tests (three or more groups). Data are shown as mean ± SEM. P values lower than 0.05 were considered statistically signifcant. Results GM-CSF induces an infammatory profle in monocytes. To examine the efect of GM-CSF on mono- cyte maturation, we stimulated freshly isolated monocytes with LPS in the presence or absence of GM-CSF for 18 h and analyzed the expression of molecules associated with monocyte maturation and antigen presentation. We found that GM-CSF induced a signifcant increase in the expression of MHC-II and CD86 compared to the PBS-treated groups. Inversely, the expression level of PDL-1 was decreased afer GM-CSF treatment. However, no signifcant diferences were found in CD80, CD83 expression between treatment and control groups (Fig. 1A,B) Given that HLA-DR and CD86 are necessary frst and second signals in antigen-presentation26, we inves- tigated whether , which are the third signal, would be afected by GM-CSF treatment. We found that GM-CSF signifcantly decreased the level of anti- infammatory cytokines like IL-27 and Il-10 (Fig. 2A,B). On the other hand we observed the increased levels of TNF-α and IL-1β in GM-CSF treated compared with PBS-treated cultures (Fig. 2A–C). Te level of IL-1β and TNF- α was found to be signifcantly increased in culture superna- tants as detected by ELISA (Fig. 2C). Of note, IL-10 and IL-27 was signifcantly reduced afer GM-CSF treatment (Fig. 2C).

Blockade of endogenous GM-CSF induces an immunomodulatory phenotype in monocytes. Our results showed that GM-CSF induced a pro-infammatory profle in monocytes, an efect that has been pre- viously described in the mouse system27. Given that monocytes also produce small amounts of GM-CSF28, we wanted to test if blockage of endoge- nous GM-CSF would afect the maturation of monocytes afer exposure to LPS. To block endogenous GM-CSF, LPS-activated monocytes were cultured in the presence of anti-GM-CSF monoclonal antibodies. Our results showed that anti-GM-CSF treatment of monocytes signifcantly reduced expression of HLA-DR and CD86 com- pared to the control group (Fig. 3A,B). Interestingly, PD-L1 expression remained unaltered (Fig. 3A,B). PD-L1 induced modulation or anergy of PD1+ cells29. Te fact that GM-CSF supplementation or blockage had little or no efect on PD-L1 expression suggests that GM-CSF plays no role in the PD-L1-PD1 signaling axis. We also found that anti-GM-CSF treatment decreased the expression of TNF-α, IL-1β compared with controls (Fig. 4A,B). Conversely, anti-GM-CSF-treated monocytes signifcantly produced more IL-10 than controls while, there was no signifcant diference in IL-27 level between groups (Fig. 4A,B). Collectively, our data show that GM-CSF induced pro-infammatory phenotype in monocytes and its blockage induced anti-infammatory cells.

CXCL-11 is suppressed by GM-CSF. Our results confirmed that GM-CSF induces the expression of antigen-presenting molecules and stimulates the production of infammatory cytokines by monocytes. We also showed that blockage of endogenous GM-CSF had the opposite efect by inducing anti-infammatory monocytes. We then sought to investigate which genes are negatively regulated by GM-CSF. We extracted RNA from mono- cytes treated with GM-CSF. Monocytes treated with PBS were used as controls. Te RNA was reverse transcribed to cDNA, and the cDNA was run in a PCR Array plate that analyzed 96 genes. Our results showed that GM-CSF altered the total gene expression levels in human monocytes (Fig. 5A). We found that CXCL-11 was among the

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Figure 2. GM-CSF treatment induces an infammatory phenotype in monocytes. Representative fow cytometry dot plots show the level of IL-27 and IL-1β. (A) Graphs indicate the percentage of cells producing IL- 27, IL-10, and IL-1β and TNF-α in the CD14+ gate. (B) Cytokine levels in culture supernatant were verifed by ELISA (C). Results are representative of three independent experiments and are expressed as mean ± SEM.

Figure 3. GM-CSF blockade decreases the expression of MHCII and co-stimulatory molecules in monocytes. (A) Representative histograms show the isotype controls (black) and the expression level of infammatory and suppressive markers afer anti-GM-CSF antibody treatment (blue). (B) Graphs summarize fow cytometry fndings. Results are presented as mean ± SEM from three independent experiments.

genes least expressed in GM-CSF-treated monocytes (Fig. 5A). Conversely, we confrmed that anti-GM-CSF treatment upregulated CXCL-11 at the gene and levels (Fig. 5B,C, respectively). CXCL-11 is involved in the generation of regulatory T cells and in the suppression of infammation30. To investigate the contribution of CXCL-11 as a modulatory chemokine of anti-GM-CSF-treated monocytes, we performed a co-culture experiment between syngeneic monocytes and naive T cells. Monocytes were treated with GM-CSF and LPS or with LPS only. To assess their ability to stimulate T cell proliferation, GM-CSF-treated monocytes were cultured with Cell Trace Violet (CTV)-labeled naïve CD4+ T cells in the presence or absence of

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Figure 4. GM-CSF blockade induces an immunomodulatory phenotype of monocytes. Representative fow cytometry dot plots (A) Graphs show the percentage of cells producing IL-27, IL-10, IL-1β and TNF-a in the CD14+ gate afer GM-CSF blockage (A). Cytokine production was measured by ELISA. (B) Results are presented as mean ± SEM from three independent experiments.

Figure 5. GM-CSF blockade promotes CXCL-11 expression. QPCR array results showed the suppression of CXCL-11 expression by GM-CSF (A). CXCL-11 expression was assessed using quantitative real-time PCR (B). Te production level of CXCL-11 was analyzed by ELISA (C). Results are presented as mean ± SEM from three independent experiments.

recombinant human CXCL-11. Afer the incubation period, cells were collected, and the dye decay was meas- ured by fow cytometry. Our data showed that while GM-CSF-treated monocytes stimulated a high proliferative response from T cells, the addition of rhCXCL-11 signifcantly suppressed this response (Fig. 6A). We also ana- lyzed the phenotype of T cells that were cultured with monocytes and found that IFN-γ production was signif- cantly suppressed in cultures conducted in the presence of rhCXCL-11 (Fig. 6B). Interestingly, the expression of FOXP3 in T cells, which is related to suppressive activity of Treg cells was increased in existence of the CXCL11 (Fig. 6C). Moreover, the T17-related transcriptional factor RORγt was considerably decreased, in T cells cul- tured in the presence of rhCXCL-11 (Fig. 6D). Tese results demonstrate that CXCL-11 is an inhibitor of T cell activation and can overcome the infammatory efect that GM-CSF exerts on monocytes.

CXCL-11 is a signifcant regulatory chemokine produced by anti-GM-CSF-treated monocytes. We found that GM-CSF directly suppresses the production of CXCL-11 while anti-GM-CSF treatment had the opposite efect (Fig. 5). We then showed that addition of rhCXCL-11 to co-cultures of GM-CSF-treated mono- cytes overcame the infammatory phenotype of monocytes and modulated T cells towards an immunoregula- tory phenotype (Fig. 6). We then investigated whether blockage of CXCL-11 would hamper the benefcial efect of anti-GM-CSF treatment on monocytes. We performed a co-culture experiment with anti-GM-CSF-treated monocytes and CTV-labeled T cells obtained from the same donors. Culture conditions included cells cultured in the presence or absence of blocking antibodies to CXCL-11. Our results showed that while anti-GM-CSF-treated monocytes had little impact on the proliferation of T cells, blockage of CXCL-11 rescued their prolifera- tion (Fig. 7A). Also, we fgured out that blockage of CXCL-11 would result in increase in the production of

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Figure 6. CXCL-11 inhibits GM-CSF-induced pro-infammatory efects of monocytes on CD4+ T cells. CFSE histograms show the rate of proliferation of T cells under various treatments. (A) Representative fow cytometry dot plots of IFN-g and GM-CSF with diferent treatments. (B) Flow cytometry Dot plots of FOXP3 expression with distinct treatments (C). Histogram analyses show the expression level of RORɤt (D). Results are presented as mean ± SEM from three independent experiments.

Figure 7. CXCL-11 is required for the immunomodulatory efects of monocytes afer GM-CSF blockade. CFSE histograms show the proliferation rate of T cells afer blockade of rhCXCL-11 and GM-CSF (A). Representative fow cytometry dot plots for IFN-γ afer blockage of CXCL-11 and GM-CSF (B). Flow cytometry Dot plots of FOXP3 expression with distinct treatments (C). Histogram analysis shows the expression level of RORɤt in diferent treatment conditions (D). Results are presented as mean ± SEM from three independent experiments.

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IFN- γ (Fig. 7B). Moreover, we found that CD4+ T cells expressed higher levels of FOXP3 when cultured with anti-GM-CSF-treated monocytes in comparison with controls, and this efect was reversed by anti-CXCL-11 (Fig. 7C). In addition, the expression of RORγt which is related to T17 cells was increased signifcantly afer neutralization of CXCL-11 (Fig. 7D). Taken together, these data confrm that CXCL-11 is a major modulatory chemokine produced by anti-GM-CSF-treated monocytes. Discussion Te signifcant infammatory role of GM-CSF in autoimmune disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA) has recently been shown3,31. Even though the mechanistic basis of the GM-CSF infam- matory role has not been fully elucidated, its biological and clinical implications are clear32–36. In this study, we show that CXCL-11 is a monocyte-derived modulatory chemokine that is suppressed explicitly by GM-CSF. We showed that GM-CSF-treated monocytes increased the expression of critical antigen-presenting molecules such as HLA-DR and CD86 as compared with the control group. Consistent with our fndings, previous studies have shown that GM-CSF primes macrophages for the produc- tion of pro-infammatory cytokines such as TNF-α and IL-6 in response to LPS37,38. In addition, Croxford et al. have indicated that GM-CSF signaling induces an infammatory signature in CCR2+Ly6Chi monocytes and their progeny, which plays a critical role in tissue destruction11. In agreement with the known mechanism of action of GM-CSF, in our study, the increase in HLA-DR and co-stimulatory molecule expression in the presence of GM-CSF and its decrease afer GM-CSF blockage suggest that the infammatory impact of GM-CSF is mediated in part through class II molecules along with co-stimulatory markers39. Next, we investigated and provided evi- dence of the role of GM-CSF blocking on T cell responses. We hypothesized that CXCL-11 would be increased afer GM-CSF blockade. We next asked whether the CXCL-11 level might afect immunomodulatory pathways of T cell responses, and we found that CXCL-11 suppressed T cell proliferation induced by GM-CSF-treated monocytes. Moreover, CXCL-11 inhibited GM-CSF-induced pro-infammatory efects of monocytes on CD4+ T cells. On the other hand, we demonstrated that CXCL-11 is involved in suppression of T cell proliferation caused by GM-CSF and also that this chemokine is required for the immunomodulatory efects of monocytes on CD4+ T cells. CXCL-11 is a chemokine that regulates cell trafcking via communication with a specifc 7- transmembrane G protein-coupled receptor (GPCR)40. Tis chemokine plays a vital role in the induction of , tis- sue extravasation, and leukocyte diferentiation41. Our fndings of increased expression of PD-L1, CD39 (data not shown), and IL-10, with a decreased level of T cell proliferation and RORγt expression in treatments with rhCXCL-11 and anti-GM-CSF, show a critical relationship between GM-CSF and CXCL-11 in infammation regulation. CXCL9 (MIG), CXCL10 (IP-10), and CXCL-11 (I-TAC) can bind to a called CXCR3, which is expressed on efector T cells, T17 and also NK cells42–46. Te binding epitope site of CXCL-9 and CXCL-10 on CXCR3 is diferent from that of CXCL-1145,46. Tese three are largely secreted by monocytes, endothelial cells, fbroblasts, and cancer cells47. Notably, CXCL-11 has a much greater afnity for binding to CXCR3 than CXCL-9 and CXCL-10, resulting in desensitization of the receptor45,46, which makes it a possible antagonist of two other ligands. In a mechanistic study, Paterka et al. assessed the role of CD11c+ cells in neuroinfammation. Tey indicated that IL-17 production by T17 cells is induced by GM-CSF, and more interaction between T17 cells and den- dritic cells locally reactivates T17 cells. Tis loop stimulates T17 cells for the production of higher levels of GM-CSF, which is required for CD11c+ cell induction48. Furthermore, the role of GM-CSF in the induction of experimental autoimmune encephalomyelitis (EAE) is not related to IL-17 and IFN-γ7,48. CXCL-10 potentially drives T1 cell diferentiation, whereas it has been suggested that CXCL-11 induces FOXP3- negative regula- tory T cells that suppress autoimmune encephalomyelitis30,49. Te robust production of CXCL10 and CXCL9 by CD11c+ cells in EAE mice and the depletion of T cell retention or accumulation in the CNS of GM-CSF-defcient (Csf2−/−) and CD11c+- depleted mice also demonstrate the infammatory efects of GM-CSF48,50. In addition, suppression of CXCL10 and CXCL-11-induced chemotaxis does not affect IL-10 and IFN-γ production by CXCL-11 and CXCL10, respectively51. Accordingly, it thus appears that CXCL-11 binding to CXCR3 regulates infammatory immune responses that occur in the absence of GM-CSF49. In summary, our fndings indicate that GM-CSF blockade not only inhibited the pro- infammatory profle of monocytes, but they also suggest an immunomodulatory role. Our study also strengthens the possibility that GM-CSF may contribute to infammatory responses through suppression of CXCL-11 production. Induction of CXCL-11 afer GM-CSF neutralization and enhanced production of immunoregulatory markers such as IL-10 and PD-L1 afer CXCL-11 treatment indicate that CXCL-11 promotes anti-infammatory responses and that GM-CSF likely interferes with that function. More studies are warranted to test if CXCL11 treatment can suppress chronic infammatory autoimmune diseases, such as MS and RA.

Received: 12 August 2019; Accepted: 7 January 2020; Published: xx xx xxxx

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